Staying Alive: Vibrio cholerae's Cycle of Environmental Survival, Transmission, and Dissemination

Abstract

Infectious diseases kill nearly 9 million people annually. Bacterial pathogens are responsible for a large proportion of these diseases, and the bacterial agents of pneumonia, diarrhea, and tuberculosis are leading causes of death and disability worldwide. Increasingly, the crucial role of nonhost environments in the life cycle of bacterial pathogens is being recognized. Heightened scrutiny has been given to the biological processes impacting pathogen dissemination and survival in the natural environment, because these processes are essential for the transmission of pathogenic bacteria to new hosts. This chapter focuses on the model environmental pathogen Vibrio cholerae to describe recent advances in our understanding of how pathogens survive between hosts and to highlight the processes necessary to support the cycle of environmental survival, transmission, and dissemination. We describe the physiological and molecular responses of V. cholerae to changing environmental conditions, focusing on its survival in aquatic reservoirs between hosts and its entry into and exit from human hosts.

Figure 1. Role of biofilms in V. cholerae survival, transmission, and dissemination

A) Biofilms play an important role in V. cholerae environmental protection, transmission into the human host, and dissemination to new hosts and back into environmental reservoirs. V. cholerae can be readily found growing in biofilms in the aquatic environment, often in association with zooplankton, phytoplankton, detritus, sediment, or oceanic chitin rain. This growth mode protects from a number of environmental stressors, including nutrient limitation and predation, and allows V. cholerae to survive in the aquatic environment year round. The manual removal of biofilms and plankton-associated biofilms from the environment has been shown decrease transmission during seasonal outbreaks. Additionally, the ingestion of V. cholerae grown in biofilms allows for the delivery of both higher numbers of bacteria and hyperinfectious cells. Though the role of biofilms during host infection is still being studied, biofilm-like aggregates have been observed in patient stool and also exhibit a hyperinfectious phenotype, suggesting that biofilms not only play a role in transmission from the environment to the host, but also in the spread of cholera from host to host. B) VpsR and VpsT are the master positive regulators of biofilm genes and positively regulate one another’s expression and genes involved in biofilm formation. VpsR additionally activates the expression of a master virulence regulator, AphA, which in turn activates VpsT expression. VpsT activity is dependent on its interaction with the small signaling molecule, c-di-GMP, which is synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). The quorum sensing regulator, HapR, represses expression of VpsR, VpsT, and AphA in response to high cell density. At low cell density, HapR is inactivated and biofilm formation is upregulated. H-NS (histone-like nucleoid structuring protein) is additional negative regulator of biofilm formation. Its repressive function is silenced by VpsT. C) An electron scanning microscopy image of a V. cholerae biofilm shows cells encased in biofilm matrix.

Figure 2. V. cholerae regulation of nutrient acquisition

V. cholerae utilizes various uptake systems to acquire nutrients from the external environment. A) During chitin utilization, chitin oligomers enter the periplasm through chitoporins in the outer membrane. Once in the periplasm, chitin oligomers bind to the chitin binding protein (CBP), allowing it to release from and relive repression of the histidine kinase (HK), ChiS, which is part of a two component system (TCS). Once active, ChiS activates an as of yet unidentified response regulator (RR), ChiR, which upregulates genes involved in chitin catabolism and utilization. While ChiS is thought to also play a role in TfoS activation, the mechanism has not been identified. However, it is known that TfoS binds to chitin oligomers in the periplasm and dimerizes to become active. Once in its active conformation, TfoS upregulates the expression of the small RNA (sRNA), tfor, which, in turn, activates translation of tfox mRNA. TfoX goes on to upregulate genes involved in competency, including the chitin regulated pilus and QstR. The activation of both the chitin catabolism and competency pathways are also dependent on the cAMP-CRP complex and in the absence of this complex are repressed. B) Glycogen storage is activated in response to nitrogen limitation. The first reaction in glycogen synthesis is catalyzed by the ADP-glucose pyrophosphorylase enzymes GlgC1 and GlgC2, which generate ADP-glucose from ATP and glucose-1-phosphate. Subsequently, the enzymes GlgA and GlgB build glycogen by forming α-1,4 and α-1,6 linkages, respectively, between ADP-glucose monomers. Glycogen breakdown is initiated by three enzymes: the glycogen debranching GlgX, the maltodextrin phosphorylase GlgP, and the 4-α-glucanotransferase MalQ. Additionally, in response to unknown environmental stimuli the TCS VarSA is activated and has been shown to enhance glycogen storage and post-transcriptionally repress the global transcriptional regulator, CsrA. C) Environmental inorganic phosphate (Pi) levels regulate a number of cell processes in V. cholerae. When Pi is high, V. cholerae initiates the biosynthesis of large amounts of inorganic polyphosphate (poly-P), composed of long chains of linked Pi, via the polyphosphate kinase, PPK. When Pi is limited the TCS PhoBR is activated and regulates a number of cellular processes, including virulence, motility, biofilm formation, and Pi uptake. D) V. cholerae uses a number of mechanisms to facilitate iron acquisition. Iron uptake is regulated by the iron-dependent regulator, Fur. When iron levels are high, Fur complexes with ferrous iron (Fur-Fe²⁺) and directly binds to conserved regions on the genome, called Fur boxes, to regulate the transcription of target genes. The Fur-Fe²⁺ complex upregulates genes involved in iron storage, metabolism, and antioxidant defense and represses iron uptake genes, including the genes encoding the Feo and Fbp transport systems, which facilitate uptake of ferrous and ferric iron, respectively. Under iron limited conditions, V. cholerae produces and secretes the siderophore vibriobactin via the VibBDEFH system. Ferric vibriobactin is imported back into the cell via the outer membrane protein ViuA and both of V. cholerae’s TonB-ExbBD complexes. Ferric vibriobactin is then transported through the periplasm to the inner membrane by the periplasmic binding protein, ViuP, and then across the inner membrane by two transport systems, ViuPDGC and VctPDGC. The cytoplasmic esterase, ViuB, processes ferric vibriobactin and removes the iron from the siderophore so that it may used within the cell. V. cholerae can import siderophores produced by other bacteria, including eneterobactin, which is recognized by two enterobactin receptors, IrgA and VctA, and then transported across the outer membrane with energy supplied by the TonB2-ExbBD complex, followed by shuttling across the inner membrane by the transport systems ViuPDGC and VctPDGC. The enzyme responsible for processing ferric enterobactin in V. cholerae has not been identified.

Figure 3. Regulation of V. cholerae Type 6 Secretion System (T6SS)

The V. cholerae T6SS plays an important role in the life cycle of this pathogen, enhancing inter- and intra-species competition, protection from predators, and virulence. In strains where this system is not constitutively active, the T6SS is regulated in response to a number of environmental signals. Though it is unknown what signal TsrA responds to, this regulator represses the T6SS and the master virulence regulator ToxT, while activating HapA expression, which is involved in mucin degradation. Low osmolarityresults in activation of the osmoregulator, OscR, which represses the T6SS. Quorum sensing (QS) also regulates the T6SS in response. At low cell density, LuxO is phosphorylated and activates the expression of quorum regulatory small RNAs (Qrr sRNAs), which repress the T6SS through both direct binding to the promoter regions of T6SS genes and through their inhibition of the positive regulator of T6SS, HapR. At high cell density, both HapR and the cAMP-CRP complex activate T6SS. HapR also actives QstR, which upregulates T6SS in response to growth on chitin. Flagellar regulatory genes are known to repress the T6SS through an unknown mechanism. Additionally, VasH, which is encoded by the T6SS pathogenicity island, is known to activate T6SS genes, potentially through its interaction with the alternative sigma factor RpoN, which appears to co-regulate T6SS genes in cAMP-CRP dependent manner. Intriguingly, RpoN is also known to active Qrr sRNAs, which repress the T6SS.

Figure 4. V. cholerae adaptation to low pH and radical nitrogen species (RNS)

After ingestion, V. cholerae must adapt to low pH and reactive nitrogen species encountered in the stomach and small intestine. CadC, a ToxR-like transcriptional regulator, mediates the acid tolerance response (ATR) and is known to be activated in response to low pH and by the LysR-type regulator AphB. CadC activates the expression of a lysine decarboxylase, CadA, which is thought to pump H+ ions out of the cell, thus raising internal pH. The glutathione synthetase, GshB, is also known to increase acid tolerance, likely through its regulation of the Kef system, which is responsible for potassium ion transport and plays a role in pH homeostasis. Low pH also contributes to the production of RNS, as acidified nitrite generated in response to low pH can be reduced to RNS. Inducible NO synthase (iNOS) produced by epithelial cells is also used to generate RNS. V. cholerae exposure to RNS can result in DNA damage that may be counteracted through the expression of RecO, a protein involved in daughter strand gap repair, Nfo, an endonuclease involved in base excision repair, and MutS, a DNA mismatch repair protein. Additionally, activation of HmpA, an enzyme responsible for destroying nitric oxide (NO), via the transcriptional regulator NorR contributes to V. cholerae resistance to RNS stress.

Figure 5. V. cholerae adaptation to host signals in the small intestine

A) In the intestinal lumen, V. cholerae encounters antimicrobial peptides and high concentrations of bile. Efflux pumps promote bile and AMP resistance by removing these compounds from the cell. Multiple porins, including OmpU and OmpT, contribute to AMP resistance. OmpU promotes bile resistance, as its relatively small channel size prevents bile compounds from entering the cell. Bile acids trigger an increase in the concentration of intracellular c-di-GMP; this response may be partially quenched by the low concentration of bicarbonate present in the intestinal lumen. Bile acids also interfere with the ability of the major virulence regulator ToxT to bind DNA, thus encumbering expression of virulence genes in the lumen. B) Upon contacting the mucus layer, V. cholerae encounters mucins and high concentrations of bicarbonate. Mucins promote production of the GlcNac-binding protein GbpA, which is expressed on the cell surface and facilitates adhesion to the mucus layer. Mucins also promote production of Hap (hemagglutinin/protease), which breaks up mucus, thus facilitating penetration through the mucus layer. Hap downregulates GbpA, which may prevent the cell from continuing to adhere to the mucus layer as it penetrates through it. Additionally, mucins promote motility and downregulate vps genes, which may further foster movement through the mucus layer toward the epithelium. High cell density leads to activation of HapR, which downregulates GbpA production directly, as well as indirectly by promoting production of Hap. Virulence genes are activated when the high concentration of bicarbonate in the mucus layer enhances ToxT activity, as well as when the bile salt taurocholate promotes activation of TcpP.

Figure 6. Regulation of V. cholerae virulence cascade and mucosal escape

A) When V. cholerae contacts host epithelial cells, expression of the phosphodiesterase VieA is upregulated, resulting in a decrease in intracellular c-di-GMP concentration. In turn, low c-di-GMP concentration induces expression of toxT, which controls the production of V. cholerae’s major virulence factors, toxin co-regulated pilus (TCP) and cholera toxin (CT). Production of TCP and CT also depends on two transmembrane transcriptional regulators, ToxR and TcpP, as well as the cytoplasmic regulators AphAB and ToxT. TCP is a type IV pilus that facilitates colonization of the intestinal epithelium, while CT is a secreted toxin that causes constitutive cyclic AMP production in host epithelial cells, leading to profuse secretion of chloride and water into the gut lumen. B) During later stages of infection, as the population density increases and cells reach stationary phase, production of the starvation/stationary phase alternative sigma factor RpoS and the quorum sensing master regulator HapR are induced. These regulators trigger flagellar assembly and chemotaxis, which foster exit from the host. Additionally, the population of V. cholerae cells in the small intestine becomes bifurcated; half of the cells continue to show high expression of virulence genes (represented by bottom cell), while the other half shows downregulation of virulence genes.